1. Field of the Invention
The present invention relates to the rapid, high-sensitivity detection of bacterial contamination in malt beverages and, in particular, to a nested polymerase chain reaction method for the amplified detection of nucleic acid sequences associated with lactic acid bacterial contamination in such beverages.
2. Description of Related Art
The process of preparing fermented malt beverages, such as, beer, ale, porter, malt liquor, and other similar fermented alcoholic beverages, hereinafter referred to simply as "beer" for convenience, is historically well established. As practiced in modern breweries, the process, in brief, comprises preparing a "mash" of malt, usually with cereal adjuncts, and heating the mash to solubilize the proteins and convert the starch into sugar and dextrins. The insoluble grains are filtered off and washed with hot water that is then combined with the soluble material. The resulting wort is boiled in a brew kettle to inactivate enzymes, sterilize the wort, extract desired hop components from added hops, and coagulate certain protein-like substances. The wort is then strained to remove spent hops and coagulum, cooled, pitched with yeast, and fermented. The fermented brew, known as "green" or "ruh" beer, is then aged ("lagered") and clarified, filtered, and carbonated to produce the desired finished beer.
During the brewing process, the brewer must constantly be on guard against contamination by bacteria that can spoil the beer. A number of methods have been devised over the years to detect these bacteria, but, until recently, these methods were laborious and time-consuming, adding to the manufacturing cost.
For example, traditional methods for detecting the presence of bacteria in comestibles require an incubation period to allow for recovery of injured bacteria, growth of these bacteria from a background of competing microorganisms, and an increase in bacterial cell numbers to more readily aid in identification. More than one incubation may be needed.
These conventional methods are slow, requiring 5 to 14 days or more, depending upon the bacteria species of interest, and may be inaccurate, providing false negatives.
Barney et al. (Tech. Q. Master Brew Assoc. Am. 29(3):91-95 (1992)) discuss the difficulties attendant the detection of slow-growing beer spoilage microorganisms, which require fairly specific conditions to support detectable growth using classical culturing techniques.
The same article, however, also points out that direct detection techniques that do not depend on culturing (for example, direct epifluorescence technique (DEFT), PCR techniques, or flow cytometry) are susceptible to interference, which causes high error rates; are labor intensive; and are difficult to automate.
Also discussed are the so-called "rapid" indirect detection methods, which require three to five days using the impedance/conductance method, spectrophotometry, or metabolite detection, or two to three days using ATP bioluminescence.
In general, the article explores the shortfalls of each of the available methodologies and emphasizes the need for improved, rapid detection methods for dealing with beer spoilage microorganisms.
New biological methods, some of which involve the polymerase chain reaction (PCR), have contributed significantly to improving the accuracy of the assay and reducing the time required to obtain useful results.
PCR is a process for amplifying nucleic acids so as to improve the sensitivity and specificity of their detection. It involves the use of two oligonucleotide primers, a polymerization agent, a target nucleic acid template, and successive cycles of denaturation of nucleic acid, annealing, and extension of the primers to produce a large number of copies of a particular nucleic acid segment. With this method, segments of single copy genomic DNA can be amplified to more than 10 million.
U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose the process as:
(1) denaturing DNA template strands at elevated temperature; PA1 (2) annealing oligonucleotide primers to the DNA templates at the 3' ends of the sequence of interest at hybridization temperature; and PA1 (3) extending the 3' ends of the primer with nucleotide triphosphates in the presence of a thermostable DNA polymerase, whereby the desired template sequence is replicated. PA1 (1) filtering the infected beer sample through a polycarbonate membrane filter; PA1 (2) ultrasonicating the filter in a volatile solvent solution; PA1 (3) isolating the lactic acid bacteria from the filter and evaporating the volatile solvent; PA1 (4) treating the residual lactic acid bacteria-containing sample with lysozyme, mutanolysin, proteinase K, and SDS to extract DNA from that sample; PA1 (5) using tRNA coprecipitant to precipitate DNA from the sample, after it has been resuspended in ethanol; PA1 (6) then, finally, applying the polymerase chain reaction to the DNA precipitate using either of two selected oligonucleotides (or one of their respective complements): PA1 enzymatically amplifying at least one fragment of said extracted DNA using at least one highly conserved primer to produce a first amplified sample; and PA1 enzymatically amplifying a product sequence from said first amplified sample, using at least one less highly conserved primer to produce a second amplified sample; and then PA1 enzymatically amplifying a universal fragment of bacterial 16S rDNA under relatively inefficient conditions using a pair of 16S rDNA specific primers to produce a first amplified sample; and PA1 further enzymatically amplifying a product sequence from said first amplified sample using a pair of lactic acid bacteria specific primers to produce efficiently a second amplified sample; and then PA1 enzymatically amplifying at least one fragment of said extracted DNA using at least one highly conserved primer to produce a first amplified sample; and PA1 enzymatically amplifying a product sequence from said first amplified sample, using at least one less highly conserved primer to produce a second amplified sample; and then PA1 (1) Those that are permanently magnetizable, or ferromagnetic; and PA1 (2) Those that demonstrate bulk magnetic behavior only when subjected to a magnetic field, generally referred to as magnetically responsive particles or superparamagnetic particles.
Steps 1 to 3 are then repeated cyclically, with the primer extension products of one cycle becoming the templates for the next.
U.S. Pat. No. 4,683,202 also discloses a nested PCR method in which a second set of primers is used to amplify a smaller DNA sequence contained within the DNA sequence amplified by the first primer set. These nested or inner primers will flank the target nucleic acid. The term "flanking primers" is used to describe primers that are complementary to segments on the 3' end portions of the double-stranded nucleic acid segment that is polymerized and amplified during the PCR process.
U.S. Pat. No. 4,965,188 discloses a process for amplifying any target nucleic acid sequence contained in a nucleic acid or mixture thereof that comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers and extending the primers with a thermostable enzyme to form complementary primer extension products that act as templates for synthesizing the desired nucleic acid sequence. The amplified sequence is said to be readily detectable. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed.
U.S. Pat. No. 5,075,216 discloses that dideoxynucleotide DNA sequencing methods can be improved by utilizing the DNA polymerase from Thermus aquaticus (Taq) to catalyze the primer extension reactions.
U.S. Pat. No. 5,079,352 discloses recombinant DNA vectors that encode a thermostable DNA polymerase and are said to be useful in the recombinant production of thermostable DNA polymerase. The recombinant thermostable polymerase is preferred for use in the production of DNA in a polymerase chain reaction. Especially useful vectors encode the about 94,000 dalton thermostable DNA polymerase from Thermus aquaticus.
U.S. Pat. No. 5,139,933 discloses an assay method to quickly detect the presence of Listeria strains in samples, characterized by the use of antibodies to selectively capture the peptidoglycan and teichoic acid components of the listeriae bacterial cell wall.
U.S. Pat. No. 5,314,809 discloses methods for enhanced specificity and sensitivity of nucleic acid amplification. The methods are simplified nested amplification procedures wherein both inner and outer primer pairs are present in the amplification reaction mixture. According to the methods, the thermocycling profile, as well as the sequences, length, and concentration of amplification primers, are modified to regulate which primers are annealed and extended on the target during any particular amplification cycle.
U.S. Pat. No. 5,340,728 discloses an improved method for performing a nested PCR amplification of a target piece of DNA, wherein by controlling the annealing times and concentration of both the outer and the inner set of primers, highly specific and efficient amplification of a targeted piece of DNA can be achieved within one reaction vessel without depletion or removal of the outer primers from the reaction mixture vessel.
U.S. Pat. No. 5,556,773 discloses a nested polymerase chain reaction performed in a single reaction tube that remains closed after the reaction mixtures for each amplification have been introduced therein. The reaction mixture for the second PCR amplification is sequestered and preserved in an upper portion of the single, closed reaction tube during the first amplification, and subsequently introduced into the reaction space containing the end product of the first PCR amplification, without opening the reaction tube.
EP 200362 and EP 201184 disclose a process for amplifying and detecting any target nucleic acid sequence contained in a nucleic acid or mixture thereof. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers to form complementary primer extension products that act as templates for synthesizing the desired nucleic acid sequence, and detecting the sequence so amplified. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired.
In addition, a specific nucleic acid sequence may be cloned into a vector by using primers to amplify the sequence, which contain restriction sites on their noncomplementary ends, and a nucleic acid fragment may be prepared from an existing shorter fragment using the amplification process.
EP 258017 discloses a purified enzyme having unique characteristics. Preferably, it is isolated from the Thermus aquaticus species and has a molecular weight of about 86,000 to 90,000 daltons. The thermostable enzyme may be native or recombinant and may be used in a temperature-cycling chain reaction wherein at least one nucleic acid sequence is amplified in quantity from an existing sequence with the aid of selected primers and nucleotide triphosphates. The amplification process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers with a thermostable enzyme to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence, and detecting the sequence so amplified. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed. The enzyme is preferably stored in a buffer of nonionic detergents that lends stability to the enzyme.
The use of polymerase chain reaction methodologies in the detection of beer spoilage infections is also known in the art. For example, in J. Am. Soc. Brew. Chem. 51(1):40-41 (1993) (see also, J. Am. Soc. Brew. Chem. 50(2):64-67 (1992)), there is disclosed a method for detecting the beer spoilage microorganism Lactobacillus brevis, in particular, using a polymerase chain reaction technique. The method requires that the sample be filtered through a submicron filter, after which the filter is ultrasonicated in an ethanol bath in order to improve the release of cells from the filter substrate. Transfer RNA (tRNA) is added to the ethanol to coprecipitate the extracted DNA and a Pfu polymerase is used in the PCR amplification process, whereby the detection limit is lowered from 30 cells to 9 cells per 250 mL of beer sample.
Japanese published application number 6141899 details a process for the highly sensitive detection of lactic acid bacteria, L. brevis in particular, as follows:
5'-TGTGGTGGCGATAGCCTGAA-3' (SEQ ID NO:1) or 5'-GCGTGGCAACGTCCTATCCT-3' (SEQ ID NO:2). PA0 Pediococcus damnosus; PA0 Lactobacillus brevis (varlindneri); PA0 Lactobacillus plantarum; PA0 Pediococcus damnosus (cerevisiae); PA0 Lactobacillus acidophilus; PA0 Lactobacillus bulgaricus; PA0 Leuconostoc mesenteroides; PA0 Lactobacillus helveticus; PA0 Pediococcus inopinatus; PA0 Lactobacillus fermentum; PA0 Lactobacillus casei alactosus; PA0 Pediococcus halophilus; PA0 Lactobacillus delbrueckii; PA0 Leuconostoc mesenteroides subsp. dextrinicum; PA0 Lactobacillus buchneri; PA0 Lactobacillus casei varcasei; PA0 Pediococcus intermedius; PA0 Pediococcus parvulus; PA0 Pediococcus dextrinicus; PA0 Streptococcus lactis.
The detection time is reportedly reduced to about 11 hours.
DiMichele et al. (J. Am. Soc. Brew. Chem. 51(2):63-66 (1993)) disclose a more rapid technique that could be carried out in about six hours. However, that technique was species specific and had a detection threshold of about 20 cells per mL in beer. The process depended on sample filtration and dissolution, followed by PCR amplification and gel electrophoresis of certain species specific regions of the 16S rRNA. While the elapsed time for the detection was reduced to six hours, samples had to be carried out in parallel for each of a predetermined number of species (for example, for L. brevis, L. casei, and L. plantarum), which makes the method very labor intensive and, hence, costly.
U.S. Pat. No. 5,484,909 discloses nucleic acid sequences that preferentially bind to the rRNA or rDNA of microorganisms that cause the spoilage of beer. The beer spoilage microorganisms are predominantly of the genera Lactobacillus and Pediococcus. The nucleic acids can be used as probes in assays to detect the presence of these microorganisms. In practice, a sample, such as a swab or liquid aliquot is processed to liberate the total nucleic acid content. The sample, putatively containing disrupted beer-spoilage organisms, is incubated in the presence of a capture probe, detector probe, and magnetic particle beads, which have been derivatized with oligo-deoxy Thymidine in chaotropic buffer such as guanidine isothiocyanate.
If target molecules (beer-spoilage microorganisms of the genus Pediococcus or Lactobacillus) are present, a Bead-Capture Probe-Target-Detector Probe hybridization complex is formed. The presence of a magnet near the bottom of the reaction tube will cause the magnetic particle-hybridization complex to adhere to the side of the tube, enabling the removal of the sample matrix, unbound probe, and other constituents not hybridized.
In Advances in Detection and Identification Methods Applicable to the Brewing Industry, in BEER AND WINE PRODUCTION (ACS Symp. Ser. 536) 13-30 (B. P. Gump ed. 1993), Dowhanick and Russell survey contemporaneous developments in techniques that are potentially useful for the detection and identification of beer spoilage microorganisms. Impedimetric detection, ATP bioluminescence, protein characterization using polyacrylamide gel electrophoresis, immunological analysis, DNA probe hybridization, karyotyping using pulsed field gel electrophoresis, are all discussed. In addition, the paper deals with DNA sequence amplification using PCR either with specifically designed probes or nonspecific "random amplified polymorphic DNA probes." While all of the described methodologies are recognized as being much faster than conventional microbiological analyses that are based on culturing and microscopic examination, they are not generally practical for routine quality control purposes.
Stewart et al. (J. Am. Soc. Brewing Chemists 54(2):78-84 (1996)) disclose a nested PCR protocol that greatly improves the sensitivity of detection of lactic acid bacteria in the presence of a high concentration of interfering substances, such as yeast cells in a fermenter sample. By using the 16S-rDNA genes as targets for PCR amplification, they taught that primers could be designed to react with a family of lactic acid bacteria that are potential beer spoiling organisms, but do not react with other nonspoilage organisms. This is possible because the rDNA contains both highly conserved sequences to all bacterial species as well as highly variable regions that are unique to individual species or families of species. The sensitivity of detection is very high, allowing application of the nested PCR protocol to the screening of samples of fermenting yeast for the presence of lactic acid bacteria. The short time required to complete the assay (six hours) allows the monitoring of yeast later in fermentation, thereby allowing an improved assessment of contamination by lactic acid bacteria. The disclosure of this article is incorporated herein by reference.
It is clear that there remains a need in the brewing arts for an efficacious method for rapidly detecting bacterial infections.